Primed templates are nucleic acids comprised of two nucleic acid strands of unequal length bound together to form a substrate for polynucleotide synthesis. Primed templates are used in a wide variety of molecular biological techniques, including gene cloning, in vitro gene mutagenesis, nucleic acid amplification, nucleic acid detection, and the like.
Primed templates are typically produced by hybridizing (annealing) a primer to a target nucleotide sequence on the template that is complementary to the sequence of the primer. The fidelity of hybridization reactions of primer with template is known to vary depending on a variety of factors, including temperature, complexity of the template, and the like. Generally, greater template length and higher hybridization temperatures each contribute to increased mismatching between primer and template resulting in inappropriately and undesirably primed template.
Inappropriately primed template is the major cause of undesirable (background) primer extension reaction products in primer extension reactions. This is particularly the case in the polymerase chain reaction (PCR) method for amplifying specific nucleic acid sequences. See, for example, Beck et al, Nuc. Acid Res., 16:9051 (1988); and Haqqi et al, Nuc. Acid Res., 16:11844 (1988).
In PCR, specific nucleic acid sequences are amplified using a chain reaction in which primer extension products are produced using primed nucleic acid templates. The product of each primer extension reaction specifically anneals with a primer and the resulting primed template acts as a substrate for further primer extension reactions. PCR is particularly useful in detecting nucleic acid sequences which are initially present in only very small amounts. However, the utility of PCR is often hampered by high levels of background primer extension reaction products due to primer/template mismatching. The procedure for conducting PCR has been extensively described. See U.S. Pat. No. 4,683,195 and No. 4,683,202 both to Mullis et al.
Single-stranded nucleic acid binding proteins (SSB) have been characterized in some detail and include such members as the E. coli single-stranded binding protein (Eco SSB), T4 gene 32 protein (T4 gp32), T4 gene 44/62 protein, T7 SSB, coliphage N4 SSB, adenovirus DNA binding protein (Ad DBP or Ad SSB), and calf thymus unwinding protein (UP1). Chase et al, Ann. Rev. Biochem., 55:103-36 (1986); Coleman et al, CRC Critical Reviews in Biochemistry, 7(3):247-289 (1980); Lindberg et al, J.Biol. Chem., 264: 12700-08, 1989; and Nakashima et al, FEBS Lett. 43: 125, 1974.
Eco SSB is an SSB that increases the fidelity of DNA replication and stimulates E. coli DNA polymerases II and III but not polymerase I or T4 DNA polymerase. Chase et al, Ann. Rev. Biochem., 55:103-36 (1986). Eco SSB has been shown to relieve pausing by DNA polymerase III assemblies at regions of secondary structure [(LaDuca et al, Biochem., 22:5177-87 (1983)] and in vitro studies of RecA-mediated reactions suggest that SSB affects ssDNA by removing secondary structures. Muniyappa et al, Proc. Natl. Acad. Sci. USA, 81:2257-61 (1984).
T4 Gene 32 protein is an SSB coded for by gene 32 of bacteriophage T4. One of its functions is to assist T4 DNA polymerase synthesis across regions of secondary structure in a single-stranded template. Huberman et al, J.Mol.Biol., 62: 39-52 (1971). It has also been used in an in vitro mutagenesis reaction to promote uninterrupted synthesis from template by addition of gene 32 protein to the template after a mutagenized primer is annealed but before polymerization. Muta-Gene M13 in vitro mutagenesis kit instructions, version #189 89-0096, p.34, Bio-Rad Laboratories, Richmond, Calif.
SSB therefore has traditionally been viewed as functioning by minimizing secondary structure in ssDNA and thereby facilitating enzyme passage (processivity) along the DNA template. In this context, SSB has been used in a variety of ways based on this property of SSB.
Zapoliski et al, in published PCT Patent Application No. WO85/05685, describe the use of Eco SSB in combination with E. coli RecA protein and ATP to form a hybridization mixture having the capacity to stimulate the transfer of ssDNA to homologous duplexes. The Zapoliski disclosure indicates that a cooperative binding between Eco SSB and the ssDNA increases the homologous pairing mediated by RecA.
Kaspar et al, Nuc. Acids Res., 17:3616 (1989) describes the use of T4 gp32 in the primer annealing and the primer extension steps of a dsDNA sequencing procedure. The Kaspar disclosure states that adding T4 gp32 allows Klenow enzyme to read through a region that previously caused termination, which suggests that the utility of T4 gp32 in a sequencing protocol was in reducing secondary structure to allow the polymerase to continue down the template.